The present invention relates generally to the purification of viruses and virus-derived vectors, including those related to alphaviruses, from biological and chemical preparations. In particular, this invention relates to methods of purification of such viruses and vectors from preparations by subjecting the preparation to chromatographic purification using an ion exchange resin or combination of an ion exchange resin step and another chromatographic process step such as size exclusion or affinity chromatography. The method provides purified viruses and vectors for use as effective vaccines and therapeutics. Moreover related methods for quantifying replicon vector preparations and verifying the replication incompetency of purified vectors are provided.
Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Twenty-six known viruses and virus subtypes have been classified within the alphavirus genus, including, Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus.
Sindbis virus is the prototype member of the Alphavirus genus of the Togaviridae family. Its replication strategy has been well characterized in a variety of cultured cells and serves as a well-accepted model for other alphaviruses. Briefly, the genome of Sindbis virus (like other alphaviruses) is an approximately 12 kb single-stranded positive-sense RNA molecule which is capped and polyadenylated, and contained within a virus-encoded capsid protein shell. The nucleocapsid is further surrounded by a host-derived lipid envelope into which two viral-specific glycoproteins, E1 and E2, are inserted and anchored to the nucleocapsid. Certain alphaviruses (e.g., SFV) also maintain an additional protein, E3, which is a cleavage product of the E2 precursor protein, PE2.
After virus particle adsorption to target cells, penetration, and uncoating of the nucleocapsid to release viral genomic RNA into the cytoplasm, the replicative process occurs via four alphaviral nonstructural proteins (nsPs), translated from the 5xe2x80x2 two-thirds of the viral genome. Synthesis of a full-length negative strand RNA, in turn, provides template for the synthesis of additional positive strand genomic RNA and an abundantly expressed 26 S subgenomic RNA, initiated internally at the junction region promoter. The alphavirus structural proteins (sPs) are translated from the subgenomic 26S RNA, which represents the 3xe2x80x2 one-third of the genome, and like the nsPs, are processed post-translationally into the individual proteins.
Several members of the alphavirus genus are being developed as xe2x80x9crepliconxe2x80x9d expression vectors for use as vaccines and therapeutics. Replicon vectors may be utilized in several formats, including DNA, RNA, and recombinant vector particles. Such replicon vectors have been derived from alphaviruses that include, for example, Sindbis virus (Xiong et al., Science 243:1188-1191,1989; Dubensky et al., J. Virol. 70:508-519,1996; Hariharan et al., J. Virol. 72:950-958, 1988; Polo et al., PNAS 96:4598-4603, 1999), Semliki Forest virus (Liljestrom, Bio/Technology 9:1356-1361, 1991; Berglund et al., Nat. Biotech. 16:562-565, 1998), and Venezuelan equine encephalitis virus (Pushko et al., Virology 239:389-401, 1997). A wide body of literature has now demonstrated efficacy of such replicon vectors for applications such as vaccines (see for example, Dubensky et al., ibid; Berglund et al., ibid; Hariharan et al., ibid, Pushko et al., ibid; Polo et al., ibid; Davis et al., J Virol. 74:371-378, 2000; Schlesinger and Dubensky, Curr Opin. Biotechnol. 10:434-439, 1999; Berglund et al., Vaccine 17:497-507, 1999).
Because of their configuration, vector replicons do not express the alphavirus structural proteins necessary for packaging into recombinant alphavirus particles (replicon particles). Thus, to generate replicon particles, these proteins must be provided in trans. Packaging may be accomplished by a variety of methods, including transient approaches such as co-transfection of in vitro transcribed replicon and defective helper RNA(s) (Liljestrom, Bio/Technology 9:1356-1361, 1991; Bredenbeek et al., J. Virol. 67:6439-6446, 1993; Frolov et al., J. Virol. 71:2819-2829, 1997; Pushko et al., Virology 239:389-401, 1997; U.S. Pat. Nos. 5,789,245 and 5,842,723) or plasmid DNA-based replicon and defective helper constructs (Dubensky et al., J. Virol. 70:508-519, 1996), as well as introduction of alphavirus replicons into stable packaging cell lines (PCL) (Polo et al., PNAS 96:4598-4603, 1999; U.S. Pat. Nos. 5,789,245, 5,842,723, and 6,015,694; PCT publications WO 9738087 and WO 9918226).
Alphavirus replicon particles produced using any of the above methodologies subsequently are harvested in the cell culture supernatants. The replicon particles then may be concentrated and partially purified using one of several published approaches, including polyethylene glycol (PEG) precipitation, ultracentrifugation, or Cellufine sulfate(trademark) ion exchange chromatography. Unfortunately, these methods do not remove a sufficient level of non-alphavirus derived protein contaminants, are not scalable, or are costly, and therefore are likely not amenable for commercial manufacture necessary of vaccine and therapeutic products.
The present invention provides methods of production and purification with utility for the large-scale manufacture of alphavirus replicon particles. Also disclosed are novel methods for quantitating vector particles in a preparation and determining the presence or absence of contaminating replication-competent virus in a preparation. Additional methods are provided for detecting the presence of packaged helper RNAs in a preparation of replicon particles. Alphavirus particles produced and characterized according to the methods described herein may be used for a variety of applications, including for example, vaccines and gene therapy.
Briefly stated, the present invention provides methods of production and purification for alphavirus replicon particles. Such replicon particles may be derived from a wide variety of alphaviruses (e.g., Semliki Forest virus, Ross River virus, Venezuelan equine encephalitis virus, Sindbis virus), and are designed to express a variety of heterologous proteins (e.g., antigens, immunostimulatory proteins, therapeutic proteins).
Within one aspect of the invention, a method of purifying alphavirus replicon particles is provided. Purification is achieved by first contacting a preparation containing alphavirus replicon particles with an ion exchange resin, under conditions and for a time sufficient to bind to the resin. Next, the portion of the preparation which is not bound to the ion exchange resin is removed from the ion exchange resin, and then the bound alphavirus replicon particles are eluted from the ion exchange resin and recovered. In one embodiment, the ion exchange resin is a tentacle ion exchange resin. In another embodiment, the tentacle ion exchange resin is a cationic exchange resin. In yet another embodiment, the tentacle ion exchange resin is an anionic exchange resin.
Within another aspect of the invention, a method of purification for alphavirus replicon particles is provided, comprising at least two chromatographic purification steps. The chromatographic purification steps are selected from the group consisting of ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and affinity chromatography. In one preferred embodiment, purification is performed using a first step of ion exchange chromatography and a second step of size exclusion chromatography.
Within another aspect of the invention, a method of producing alphavirus replicon particles is provided. Alphavirus packaging cells are infected with a seed stock of alphavirus replicon particles and then incubated in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus replicon particles. Next the culture supernatants containing the replicon particles are harvested. In one embodiment, the bioreactor is an external component bioreactor. In another embodiment the bioreactor is a suspension culture bioreactor.
Within another aspect of the invention, a method of producing alphavirus replicon particles is provided. Alphavirus packaging cells are transfected with a DNA-based alphavirus replicon or eukaryotic layered vector initiation system and then incubated in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus replicon particles. Next the culture supernatants containing the replicon particles are harvested.
Within another aspect of the invention, a method of producing alphavirus replicon particles is provided wherein alphavirus packaging cells are transfected with an alphavirus RNA vector replicon transcribed in vitro and then incubated in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus replicon particles. Next the culture supernatants containing the replicon particles are harvested.
Within other aspects of the invention, methods of generating alphavirus replicon particles for use in vaccine or therapeutic applications are provided. Replicon particles are produced in packaging cell lines and purified by a chromatographic purification process as described above. In preferred embodiments, the chromatographic purification process includes a step of ion exchange chromatography using a tentacle ion exchange resin.
Within yet other aspects of the present invention, a preparation of alphavirus replicon particles is provided, wherein the preparation of replicon particles is purified by a chromatographic purification process as described above. In preferred embodiments, the chromatographic purification process includes a step of ion exchange chromatography using a tentacle ion exchange resin.
Within a related aspect, a vaccine or immunogenic composition comprising a preparation of alphavirus replicon particles purified by a chromatographic purification process as described above is provided. The preparation of replicon particles being capable of expressing an antigen derived from a pathogenic agent. In preferred embodiments, the chromatographic purification process includes a step of ion exchange chromatography using a tentacle ion exchange resin. In one embodiment, the antigen is derived from a tumor cell. In another embodiment, the antigen is derived from an infectious agent (e.g., virus, bacteria, fungus, and parasite). In preferred embodiments, the antigen is derived from HIV (e.g. gag, gp120, gp140, gp160, pol, rev, tat, and nef) or HCV (e.g. C, E1, E2, NS3, NS4, and NS5).
Within yet other related aspects, methods for stimulating an immune response within a warm-blooded animal, comprising the step of administering to a warm-blooded animal a preparation of alphavirus replicon particles purified by a chromatographic purification process as described above are provided, the preparation of replicon particles being capable of expressing an antigen derived from a pathogenic agent. In preferred embodiments, the chromatographic purification process includes a step of ion exchange chromatography using a tentacle ion exchange resin. In one embodiment, the antigen is derived from a tumor cell. In another embodiment, the antigen is derived from an infectious agent (e.g., virus, bacteria, fungus, parasite). In preferred embodiments, the antigen is derived from HIV or HCV.
Within yet other related aspects, methods for stimulating an immune response within a warm-blooded animal, comprising the step of administering to a warm-blooded animal a preparation of alphavirus replicon particles purified by a chromatographic purification process as described above are provided, the preparation of replicon particles being capable of expressing a lymphokine, cytokine, or chemokine. In preferred embodiments, the chromatographic purification process includes a step of ion exchange chromatography using a tentacle ion exchange resin. In one embodiment, the lymphokine, cytokine or chemokine is selected from the group consisting of IL-2, IL-10, IL-12, gamma interferon, GM-CSF, MIP3xcex1, MIP3xcex2, and SLC.
Still other embodiments of the present invention provide for techniques used to establish vector particle preparation safety and potency. One important aspect of vector particle safety is that the preparation be free of contaminating replication-competent alphaviral particles. The packaging cell lines used to produce the vector particles of the present invention contain at least three separate nucleic acid sources used to produce the vector particles of the present invention. One nucleic acid source contains nonstructural viral proteins and a gene of interest, another contains genes encoding for structural proteins and a third encodes for structural proteins not present in any other nucleic acid source. Therefore, contaminating replication-competent alphaviral particles can only arise if a minimum of two recombination events occur.
In one embodiment a preparation of replicon particles free from detectable contaminating replication-competent alphaviral particles is assured using polymerase chain reaction (PCR) techniques wherein a nucleic acid substrate suitable for detecting multiple recombination events is provided. The substrate is derived from a population of alphavirus replicon particles and the nucleic acid substrate is reacted with at least one first reaction mixture comprising an oligonucleotide complementary to an alphavirus nonstructural protein gene and an oligonucleotide complementary to an alphavirus structural protein gene. The structural protein gene is either a capsid protein gene or a non-capsid structural protein gene. Suitable reaction conditions and time are provided to permit amplification of the nucleic acid substrate and the formation of a first reaction product. Next, the first reaction product is reacted with a second reaction mixture containing an oligonucleotide complementary to an alphavirus capsid protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene. Suitable reaction conditions and time are provided to permit amplification of the nucleic acid substrate and the formation of a second reaction product. After the first and second reactions are complete, the presence or absence of the second reaction product is established.
In another embodiment of the present invention multiple recombination events are detected by providing a nucleic acid substrate suitable for detecting multiple recombination events, the substrate being derived from a population of alphavirus replicon particles. Then reacting the nucleic acid substrate with a first reaction mixture comprising an oligonucleotide complementary to an alphavirus nonstructural protein gene and an oligonucleotide complementary to an alphavirus capsid protein gene. Conditions suitable and for a time sufficient to permit amplification of the nucleic acid substrate to form a first reaction product are provided. Next, the first reaction product is reacted with a second reaction mixture comprising an oligonucleotide complementary to an alphavirus capsid protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene. Again, under conditions suitable and for a time sufficient to permit amplification of the nucleic acid template to form a second reaction product. Finally, determining the presence or absence of the second reaction product
In yet another embodiment a method for detecting multiple recombination events is provided comprising providing a nucleic acid substrate suitable for detecting multiple recombination events. The substrate is derived from a population of alphavirus replicon particles and then reacting the nucleic acid substrate with a first reaction mixture comprising an oligonucleotide complementary to an alphavirus nonstructural protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene. Suitable reaction conditions and time are provided to permit amplification of the nucleic acid substrate to form a first reaction product. Next the first reaction product is reacted with a second reaction mixture comprising an oligonucleotide complementary to an alphavirus capsid protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene. After a suitable incubation time, the presence or absence of the second reaction product is determined.
In one preferred embodiment, at least two of the above methods for detecting multiple recombination events are performed using the same nucleic acid substrate derived from a population of alphavirus replicon particles.
In another embodiment of the present invention replicon particle preparation potency is quantified. In this embodiment, methods are provided for quantitating or xe2x80x9ctiteringxe2x80x9d replication incompetent RNA virus vector particles in a sample. The methods comprising providing a population of packaging cells, contacting the packaging cells with the sample under conditions suitable and for a time sufficient for the cells to be infected with replication-incompetent virus vector particles. Then incubating the infected packaging cells under conditions suitable and for a time sufficient for production of virus vector particles and enumerating the number of resulting plaques.
These and other aspects and embodiments of the invention will become evident upon reference to the following detailed description and attached figures. In addition, various references are set forth herein that describe in more detail certain procedures or compositions (e.g., plasmids, sequences, etc.), and are therefore incorporated by reference in their entirety as if each were individually noted for incorporation.